Bi 150 Lecture 5

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Reminder Henry Lesters office hours outside
the Red Door Mon, 1-130 PM, Fri 2-230 PM
Bi 150 Lecture 5 Friday, October 8, 2008 Revised
10/8 Postsynaptic receptors
Chapter 11
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Synaptic transmission
Transmitter release requires influx of Ca2 into
the presynaptic terminal (through
voltage-dependent Ca2 channels)
Release is quantal. Packets of transmitter
appear to be released following binomial or
Poisson statistics.
Transmitter diffuses within the synaptic cleft
and binds to ligand-gated channels on the
postsynaptic terminal.
Opening of the ligand-gated channel produces a
postsynaptic potential, either excitatory (epsp)
or inhibitory (ipsp).
A suprathreshold epsp triggers a regenerative
impulse (action potential) that travels down the
axon of the postsynaptic neuron.
Figure 14-4
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Endplate Potential is not Regenerative (contrast
to a.p.)
The current source for the epp is restricted to
the endplate, so the size of the potential decays
quickly with distance from the endplate.
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Past lectures
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Ligand-gated ion channels
1. Multimeric integral membrane proteins

• 2. Ion flux through the channels can send a
  signal in two ways
• Change in membrane voltage
• Ca2 entry - critical for transmitter release,
  for synaptic plasticity, etc

3. Channel opens within a few µs after ligand
binding
4. Ion Selectivity Cationic, often
non-selective (Na, K, Ca2) Cl- (GABA and
Glycine receptors)
5. Subject to modulation in various ways
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From Lecture 4
Many basic principles of chemical transmission
were discovered at the neuromuscular junction
(nerve-muscle synapse, endplate) acetylcholine
is the transmitter.
Figure 11-1
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From Lecture 4
Fine structure of the NMJ
ACh receptors
Figure 11-1
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Three sources for structural information about
nicotinic ACh receptors
electric organ of electric rays, Torpedo sp (1960)
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Arrangement of subunits in the muscle nicotinic
ACh receptor
Karlin Nature Rev. Neurosci. 3 102 (2002)
Keramidas et al., Prog. Biophys. Mol. Biol. 86
161 (2004)
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Nicotinic Acetylcholine Receptor (Unwin, 2005)
2200 amino acids in 5 chains (subunits),
MW 2.5 x 106
Binding region
Membrane region
Colored by secondary structure
Colored by subunit (chain)
Cytosolic region
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The AChBP interfacial aromatic box occupied by
nicotine (Sixma, 2004) Showing the cation-p
interaction
aY198 C2
aW149 B
aY93 A
non-aW55 D
aY190 C1
(Muscle Nicotinic numbering)
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Unwins swivel hypothesis for nAChR gating
Ligand-binding domain
T
S
18'
I
I
L
L
L
L
L
L
F
F
13'
V
V
M1
T
T
L
L
M2
10'
S
A
9'
L
L
L
M3
L
L
V
V
6'
S
S
I
I
C
C
M4
L
L
2'
T
T
Intracellular loop
Miyazawa, Fujiyoshi, Unwin, Nature 2003
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The permeant ions experience a water-like
environment
Polar (Ser or Thr) OH side chains (charge
selectivity occurs in gt 2 regions)
Hydrophobic Leu (Gate)
Modified from Zigmond et al. (Eds.) Fundamental
Neuroscience, Sinauer (1999)
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Na and K ions carry the synaptic current

• Can get information by determining the Reversal
  Potential for the synaptic potential (or current).

• Erev is defined as the potential at which the
  current through open receptors is zero. Negative
  to Erev, current flows inward. Positive to Erev,
  current flows outward.
• Erevs for epps are from zero to - 15 mV.

• C. ACh receptor allows flux of both Na and K,
  and in some cases Ca2.

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Acetylcholine-induced currents have a reversal
potential near zero. The nicotinic receptor is
permeable to Na and K, but not to Cl-
Muscle nAChR
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The family of nicotinic ACh Receptors
In addition to the ???????? and ? subunits of the
muscle nicotinic receptors, the mammalian genome
contains 9 ? subunits (?2 through 10) and 3 ?
subunits (?2 through 4) that are found in
nicotinic receptors on neurons.
Nicotinic receptors always contain at least two ?
subunits. The stoichiometry of muscle subunits
is aabgd The stoichiometry of brains subunits
is sometimes (a7)5 The stoichiometry of brain
subunits is usually aabbb
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At this resolution, all Cys-loop receptors
resemble nicotinic acetylcholine receptors
2200 amino acids in 5 chains (subunits),
MW 2.5 x 106
Binding region
Membrane region
Colored by secondary structure
Colored by subunit (chain)
Cytosolic region
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Superfamilies of ligand-gated ion channels that
are synaptic receptors
Many Vertebrate Glutamate Receptors (discussed
by M Kennedy, next week) AMPA-type Kainate-type
NMDA-type
Cys-loop Receptors Nicotinic ACh Serotonin 5-HT3
GABAA and GABAC Glycine Invertebrate GluCl,
dopamine, tyrosine
ATP (P2X) Receptors
Chapter 6
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Neuronal Engineering with Cys-loop Receptor
Channels
Goal develop a general technique to selectively
and reversibly silence or activate specific
sets of neurons in vivo.
Ideal approach would Have on- and off- kinetics
on a time scale of minutes Have simple activation
(ie, via drug injected or in animals diet) Avoid
nonspecific effects in animal Maintain target
neurons healthy in an off-state for a few days
without morphological/other changes Silence or
activate diffuse molecularly defined sets of
neurons, not just spatially defined groups
The chosen channel Cys-loop receptor (like
nicotinic receptors)
Heteropentamer a2ß3 or a3ß2 subunits.
This feature allows one to intersect two
promoters, to enhance cellular specificity
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The drugs avermectins

• IVM Lactone originally isolated from
  Streptomyces avermitilis
• AVMs are used as antiparasitics in animals and
  humans (River blindness / Heartgard)
• IVM is probably an allosteric activator of GluCl
  channels
• Also modulates GABA, 5HT3, P2X, and nicotinic
  channels, at much higher doses

(IVM)
Nobel Peace Prize to Jimmy Carter, 2002
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IVM-induced silencing in GluCl-expressing
cultured rat hippocampal neurons
5 nm IVM
500 nm IVM
50 nm IVM
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We eliminated glutamate sensitivity in GluCl by
mutating a conserved aromatic residue
Colored by subunit (chain)
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AAV-2 constructs injected into mouse striatum
slice experiments Single neurons correlation
between IVM-induced conductance AP silencing
Lerchner et al, 2007 (collaboration with D. J.
Anderson at Caltech)
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Plans to extend the AVMR system
M3-M4 loop
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The first AVMR-Na
GluCl ? WT ? WT
Muscle nAChR
(10 nM IVM)
GluCl ? P(-2)?/A(-1)E ? WT
(200 nM IVM)
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Reminder Henry Lesters office hours outside
the Red Door Mon, 1-130 PM, Fri 2-230 PM
End of Lecture 5